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1. Field of the Invention
This invention relates generally to immunoglobulin testing methods and more particularly to a method of determining the concentration of IgG antibodies in biological fluids of mammals.
2. Description of the Related Art
The numbers in brackets refer to the publications listed in the Appendix, the teachings of which are incorporated herein by reference.
The bovine immunoglobulin system closely resembles that of other species with respect to physiochemical properties and nomenclature. Well characterized classes include IgG, IgM [13],IgA [14] and IgE [15-17]. The IgG class contains two documented subclasses, IgG1 and IgG2, which have antigenic differences in the Fc portion of the heavy chain. A possible third subclass, IgG3, has been reported [18], but remains unconfirmed. It is presumed that additional heterogeneity awaits discovery.
In cattle, the newborn calves are virtually devoid of any detectable levels of immunoglobulin [19, 20]. This is due to a syndesmochorial type placental attachment, which restricts the transfer of maternal immunoglobulins to fetal blood. Essentially, the neonatal calf relies entirely on passive immunity for it""s immune protection during the first weeks of life. This transfer of IgG is important in reducing mortality in calves [1, 2, 4]. Stress from birthing, temperature, or herd management practices are factors that may adversely affect the passive transfer of IgG [7-8].
Frequently, colostral supplements are included in calf rations to ensure that adequate amounts of IgG are provided [3, 5, 6]. These supplements include either frozen or natural colostrum, and/or commercial supplements. Although feeding fresh or stored colostrum seems simple, obtaining and storing adequate amounts of quality colostrum is often difficult and laborious. Additionally, the high cost of commercial colostrum prohibits its use on a routine basis. From a herd management aspect, there would be a tremendous time and monetary savings if only those animals in need of the colostral supplement could be identified quickly and accurately.
In order to assess the passive transfer of maternal immunoglobulin, serum IgG concentrations of neonatal calves may be measured. Historically, the measurement of bovine serum IgG has been performed by several methods. These methods can be divided into three main groups [21]:
a.) Accurate tests for immunoglobulin, which may or may not provide a rapid result. For these methods, blood samples must be shipped to, and analyzed in the laboratory and require professional training and equipment [12,20].
b.) Tests which can be done in a relatively short time on the farm and under xe2x80x9cfieldxe2x80x9d conditions, and do not require much training or equipment. These tests however are qualitative in nature and are not accurate. This group includes the glutaraldehyde test, the zinc sulfate turbidity test and the determination of total protein by refractometry.
c.) Tests based upon proteins other than IgG. This includes testing for xcex3-glutamyltransferase, a serum enzyme whose concentration has been reported to correlate with total globulin levels [21]. This method has never been well accepted. It is generally recommended that calves receive a minimal dose of 100 g of immunoglobulin at birth [9,10]. Calf serum IgG concentrations greater than or equal to 10 mg/ml indicates adequate passive transfer [10,11]. Values less than 10 mg/ml are considered to be indicative of Failure of Passive Transfer (FPT). It is widely accepted that the treatment for FPT is to administer two feedings of colostrum (50 g IgG/L), one within the first 24 hours of birth and the second 12 hours later. Treatment at times outside this window tend to be less effective due to the decrease in permeability of the calf intestine to ingested antibodies.
Those concerned with these and other problems recognize the need for fast, accurate and simple assay for IgG.
The present invention provides a method of determining the concentration of IgG antibodies in the biological fluids of mammals. The assay may be performed in a lateral flow cassette or dipstick format where dehydrated immobilized reagents are spaced along a membrane. A sample of a mammalian biological fluid is exposed first to an IgG complexing agent to yield a conjugate. The conjugate then moves along the membrane and is exposed to a standard mammalian IgG applied to the membrane at a test position. Binding of the IgG is indicated by a color change at the test position on the membrane. A control reagent of dehydrated anti-IgG complexing agent may be applied to the membrane at the control position spaced downstream from the test position. Interaction of the IgG complexing agent with the anti-IgG complexing agent forms a visible line at the control position on the membrane.
Therefore, an object of the present invention is the provision of an improved IgG antibody testing method.